As a powerful means for analyzing expression, mutation, polymorphism or the like of a gene, DNA chips (or DNA micro-arrays) have been proposed, and some DNA chips are put into practical use. As for a method for the preparation of DNA chips, such methods are known that surface of a solid support is previously processed to make the surface obtain plus charge, then the DNA is directly fixed to the solid support electrostatically; that synthesized oligonucleotides are fixed to the surface of the solid support via a covalent bond; or that DNA is directly synthesized on the surface of the solid support. However, these methods have merits and demerits, and an appropriate one is utilized depending on its purpose of use.
In the case where a DNA fragment to be fixed is cDNA (complementary DNA synthesized by utilizing mRNA as a template) or PCR products (DNA fragment that is amplified from the cDNA by a PCR method), in general, cDNAs or PCR products are dotted to the surface of the solid support of which the surface is treated with a polycationic compound (poly-lysine, polyethyleneimine or the like) by using a spotting device provided in a DNA chip preparation device, and are electrostatically bound to the solid support by utilizing the charge of DNA.
However, in these micro-arrays of an electrostatically bound type, an oligonucleotide of a short chain is released from the solid support at higher temperatures since the bonding force to the support is increased in proportion to length of a DNA to be fixed.
Therefore, in the case where a DNA fragment to be fixed is a synthesized oligonucleotide, an oligonucleotide into which a reactive group has been introduced is synthesized, then the oligonucleotide is dotted to the surface of the surface-treated solid support and is bound thereto via covalent bond (“Protein, Nucleic Acid, Enzyme” Vol. 43 (1998) 2004–2011; Lamture, J. B et al., Nucl. Acids Res., 22, 2121–2125, 1994; Guo, Z. et al., Nucl. Acids Res., 22, 5456–5465, 1994). For example, a method is known in which an amino group-introduced oligonucleotide is reacted with the slide glass to which an amino group has been introduced, in the presence of PDC (p-phenylene diisothiocyanate), and another method is known in which an aldehyde group-introduced oligonucleotide is reacted with said slide glass. By using these two methods, an oligonucleotide is stably fixed to the surface of the solid support, as compared with the above-described method which utilizes charge of the DNA.
The slide glass to which an oligonucleotide is fixed is often used to detect mutation or polymorphism of the gene. An analyte used for analysis of mutation or polymorphism is often selected by amplifying a region containing a polymorphism portion by PCR reaction from a genome DNA which is a sample. However, the operation in this method was complicated since it requires two steps of reaction; that is, the PCR reaction and a subsequent hybridization reaction.